Affinity-matured antibody with a disulfide bond in H-CDR3 loop

Area of Science:

• Immunology
• Structural Biology
• Biochemistry

Background:

• Antibody affinity maturation enhances antigen binding through somatic hypermutation.
• Highly matured anti-NP antibodies like E11 feature Cys96H and Cys100H in the heavy chain's CDR3, potentially forming a disulfide bond.

Purpose of the Study:

• To investigate the role of disulfide bonds in antigen binding by evaluating E11 scFv and its Cys mutants.
• To determine the thermodynamic and kinetic parameters of antigen binding for these antibodies.

Main Methods:

• Generation of single-chain Fv (scFv) antibodies, including E11 and its cysteine mutants (E11_C96KH/C100EH, E11_C96KH/C100QH).
• Determination of antigen-binding thermodynamics and kinetics.
• High-resolution crystal structure determination of NP-free and NP-bound E11 scFvs.
• Small-angle X-ray scattering (SAXS) analysis.

Main Results:

• Cysteine mutants exhibited lower binding affinities compared to wild-type E11 scFv, indicating the disulfide bond's contribution to antigen binding.
• E11 scFv affinity decreased in the presence of a reducing agent, further supporting the disulfide bond's importance.
• Crystal structures revealed the disulfide bond and elucidated the antigen recognition mechanism, allowing comparison with other anti-NP antibodies.
• SAXS data showed antigen binding induces local conformational changes in E11 scFv in solution.

Conclusions:

• The disulfide bond between Cys96H and Cys100H significantly contributes to the antigen-binding affinity and stable complex formation of E11 scFv.
• Structural insights explain the molecular basis for altered antigen-binding affinity and thermal stability.
• Antigen binding induces conformational flexibility in the antibody, influencing binding dynamics.